HPLC as a Discontinuous Method

Within the framework of the scheme just described, the HPLC method would be classified as discontinuous, since a separation step is part of the procedure. However, because termination can be accomplished by injecting the sample directly onto the column, the HPLC detection is usually on-line, that is, carried out continuously with separation. Thus, the separation and detection steps merge into a single operation, which for all practical considerations means that it is a continuous method. 

In addition, unlike many other discontinuous assays that focus on only one of the components of the reaction, the HPLC assay offers the potential to monitor several. For example, consider adenosine kinase, the enzyme that uses two substrates and forms two products according to the reaction Ado + ATP — AMP + ADP. Since HPLC can readily separate all four compounds (see Fig. 1.4), and all four compounds can be detected at 2S4 nm, it is apparent that with the HPLC method, the level of each component can be monitored during the course of the reaction, providing a complete analysis of each time point.  

In contrast, with a well-designed HPLC assay for activity E-l, capable of separating A, B, and C, the levels of each may be obtained from an analysis of a single sample from the reaction mixture of both the crude and purified samples (panel III of Fig. 1.5). In fact, the data obtained provide information not only on what compounds are present but also on what are not. The availability of such negative information can provide the data leading to the exclusion of alternative explanations that had been proposed to explain unexpected results. 

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